Evaluation of intracellular survival of Campylobacter fetus subsp. fetus in bovine endometrial cells by qPCR

Abstract

Background: Campylobacter fetus subsp. fetus is the causal agent of sporadic abortion and infertility in bovines that produces economic losses in livestock.

Aims: This study evaluates the capability of C. fetus subsp. fetus to invade and survive in bovine endometrial epithelial cells and attempts to describe a pathogenic mechanism of this microorganism.

Methods: Primary culture of bovine endometrial epithelial cells was challenged with C. fetus subsp. fetus. Intracellular bacteria, represented by the number of genomic copies (g.c.) were quantified at 0, 2, 4, 10, and 24 hours post-infection (h.p.i.), by quantitative polymerase chain reaction (qPCR). The presence of intracellular bacteria was evaluated by immunofluorescence and immunohistochemistry.

Results: The results showed that only viable C. fetus subsp. fetus could invade endometrial cells. The g.c. number in assays with viable C. fetus subsp. fetus reached an average value of 656 g.c., remained constant until 4 h.p.i., then decreased to 100 g.c, at 24 h.p.i. In assays with non-viable microorganisms, the average value of g.c. was less than 1 g.c. and never changed. The intracellular presence of this bacteria was confirmed at 2 h.p.i. by immunofluorescence and immunohistochemistry.

Conclusion: The results suggest that only C. fetus subsp. fetus viable can invade bovine endometrial epithelial cells but will not replicate in them, indicating that the endometrial cells do not represent a replication niche for this pathogen. Nonetheless, this invasion capability suggests that this type of cell could be employed by the pathogen to spread to other tissues.

Keywords: Bovine endometrial cells; Campylobacter fetus; Intracellular survival.

Fig. 1

Bovine endometrial epithelial cells cultures. (A) Normal appearance of epithelial-like cells after 7th passaging (×20), (B) Appearance of epithelial cells of bovine endometrium at 10th passaging (×20), and (C) Immunofluorescence assay of bovine endometrial cells stained with Alexa 488 (green) showed the presence of Cytokeratin 18 (×40)

Fig. 2

Intracellular survival assays. The bovine endometrial epithelial cells were infected with C. fetus ATCC 27374, S. typhimurium, and C. fetus subsp. fetus non-viable (heat-inactivated). Intracellular bacteria were quantified by qPCR at 0, 2, 4, 10, and 24 h.p.i

Fig. 3

Immunohistochemistry of intracellular C. fetus ATCC 27374 in bovine endometrial epithelial cells (×40). The microorganisms observed inside the cell are in brown and the cells are in blue color. The black arrows show the intracellular bacteria

Fig. 4

Immunofluorescence of intracellular C. fetus in bovine endometrial epithelial cells. The cytoskeleton was stained with phalloidin-FITC (green), nucleus with propidium iodide, and intracellular microorganisms with Alexa 594 (red). The white arrow shows C. fetus inside the cells